Protein fragments containing Factor VIII binding domain of von Willebrand Factor

ABSTRACT

Peptides which inhibit the binding of von Willebrand Factor to Factor VIII. Monoclonal antibodies capable of specifically binding to the region of von Willebrand Factor containing the Factor VIII binding domain. Improved methods of preparing Factor VIII.

BACKGROUND OF THE INVENTION

This invention relates to peptides which inhibit the binding of vonWillebrand factor (vWF) to Factor VIII (FVIII).

vWF and FVIII both have important but different functions in themaintenance of hemostasis. vWF participates in platelet-vessel wallinteractions at the site of vascular injury whereas FVIII acceleratesthe activation of Factor X by Factor IXa in the presence of plateletsand calcium ions. vWF and FVIII circulate in plasma as a noncovalentlylinked complex thought to be held together by both electrostatic andhydrophobic forces. vWF is thought to stabilize FVIII in vitro andprolong its half-life in the circulation. Consequently, in the absenceof endogeneous vWF the circulating half-life of FVIII is markedlyreduced. Since FVIII participates in the intrinsic pathway of bloodcoagulation, agents capable of interfering with the interaction of FVIIIand vWF would alter the FVIII level in plasma and in this manner serveas anti-thrombotic agents. The peptides of the present invention havethe ability to act as anti-thrombotic agents by their prevention of thebinding of vWF to FVIII. They also have the ability to stabilize FVIIIin an in vitro environment in which FVIII is being produced.

SUMMARY OF THE INVENTION

The present invention comprises a 29 kDa polypeptide fragment selectedfrom the following sequence: ##STR1## which inhibits binding of vonWillebrand Factor to Factor VIII, whose amino acid sequence is that of afragment of von Willebrand Factor and reacts with a monoclonal anti-vWFantibody C3 deposited with the American Type Culture Collection, 12301Parklawn Drive, Rockville, Md., 20852 with the designation (ATCC No. HB9425) capable of specifically binding to the region of von WillebrandFactor containing the Factor VIII binding domain.

Particularly preferred is a polypeptide which inhibits binding of vonWillebrand Factor to Factor VIII wherein the polypeptide has theamino-terminal sequence beginning with amino-terminal amino acid residue3 Ser and ending approximately with carboxy-terminal amino acid residue244 Leu.

Additionally preferred is a polypeptide which inhibits binding of vonWillebrand Factor to Factor VIII wherein the polypeptide has theamino-terminal sequence beginning with amino-terminal amino acid residue24 Glu and ending approximately with carboxy-terminal amino acid residue265 Ser.

Additionally preferred is a polypeptide which inhibits binding of vonWillebrand Factor to Factor VIII wherein the polypeptide has theamino-terminal sequence beginning with amino-terminal acid residue 44Gly and ending approximately with carboxy-terminal amino acid residue285 Asn.

The invention further comprises a peptide comprising a sequential subsetof at least three amino acid residues of a polypeptide fragment whichinhibits binding of von Willebrand Factor to Factor VIII and reacts witha monoclonal anti-vWF antibody C3 capable of specifically binding to theregion of von Willebrand Factor containing the Factor VIII bindingdomain and which has the following sequence: ##STR2##

The invention further comprises a new mouse-mouse hybridoma cell linewhich provides as a component of the supernatant of its growth amonoclonal anti-vWF antibody C3 capable of specifically binding to theregion of von Willebrand Factor containing the Factor VIII bindingdomain.

The invention further comprises a monoclonal anti-vWF antibody capableof specifically binding to the region of von Willebrand Factorcontaining the Factor VIII binding domain.

The invention further comprises an improved method of preparing FactorVIII by the addition of a polypeptide fragment and any sequential subsetof at least three amino acids of the polypeptide fragment which inhibitbinding of von Willebrand Factor to Factor VIII.

The invention further comprises an improved method of preparing FactorVIII using particles bound to a polypeptide fragment and any sequentialsubset of at least three amino acids of the polypeptide fragment whichinhibit binding of von Willebrand Factor to Factor VIII.

The invention further comprises a method of preparing by recombinant DNAor synthetic peptide techniques a polypeptide fragment and anysequential subset of at least three amino acids of the polypeptidefragment which inhibit binding of von Willebrand Factor to Factor VIII.

The invention further comprises an improved method for expressingrecombinant DNA produced Factor VIII using a polypeptide fragment andany sequential subset of at least three amino acids of the polypeptidefragment which inhibit binding of von Willebrand Factor to Factor VIII.

DETAILED DESCRIPTION OF THE INVENTION

As indicated the present invention encompasses polypeptide fragments andsynthetic peptides which inhibit binding of vWF to FVIII, whose aminoacid sequences are that of fragments of vWF and react with a monoclonalanti-vWF antibody C3 capable of specifically binding to the region ofvWF containing the FVIII binding domain.

The monoclonal anti-vWF antibody C3 was found to have the ability toblock the binding of purified human FVIII to purified human vWF in acrossed immunoelectrophoresis system. The epitope of C3 must resideclose to that of the FVIII binding domain of vWF. The C3 antibody wastherefore used as a marker of the FVIII binding domain.

Whole unreduced ¹²⁵ I-labeled vWF was treated with subtilisin at a 1/25(w/w) ratio for 24 hours at room temperature. This reaction mixture wasthen placed in microtiter wells which had previously been coated withmonoclonal anti-vWF antibody C3. The wells were thoroughly washed andthen treated with SDS buffer heated to approximately 90° C. and thesolution run on a 5-15% gradient SDS-PAGE gel. An autoradiograph of theSDS-PAGE gel demonstrated predominately a single band with a molecularweight of approximately 29 kDa. A similar digest of unlabeled vWF wasmade and this reaction mixture was placed on chromatography column madeup of monoclonal anti-vWF antibody C3 coupled to Sepharose 4B. The C3reactive fragments were then eluted with 3M NaSCN, dialyzed, andconcentrated. A band reactive with C3 by immunoblotting techniques wasidentified. Amino acid sequencing of this band revealed thatapproximately 60% of the amino-termini began with amino acid residuenumber 44 of the mature vWF subunit, approximately 20% began withresidue number 24 and approximately 10% began with residue number 3.

The above described experiment localized the C3 epitope and indirectlythe FVIII binding domain to the amino-terminal region of vWF. Since themolecular weight of the peptide so identified was approximately 29 kDaand its predominant amino-terminus was amino acid residue 44 of themature subunit, then the carboxy-terminus should be approximately atamino acid residue 285 based on an average molecular weight per aminoacid residue of approximately 120. Based on the published amino acidsequence of vWF in Titani et al., Biochemistry 25, 3174-3184 (1986) itis possible to synthesize peptides from the region beginning withresidue 3 and ending with amino acid residue 285 which comprises theregion of vWF containing the FVIII binding domain.

In Titani et al. the sequence analysis identified both Ala, and Thr at amolar ratio of about 4:1 at residue 26. In contrast, the nucleotidesequence of the lambda HvWF1 clone predicted Thr at residue 26 accordingto Sadler et al., Proc. Natl. Acad. Sci. USA 82, 6394-6398 (1985). Thisdiscrepancy can be due to polymorphism in the protein or to an error incDNA replication during the preparation of the DNA library. In view ofthis uncertainty at residue 26, the amino acid at residue 26, isidentified by X which represents an undetermined amino acid. Thesepeptides can interfere with FVIII-vWF interaction and thus serve asantithrombotic agents. Additional monoclonal antibodies to this regioncan be produced which will also interfere with FVIII-vWF interaction andthus can also serve as anti-thrombotic agents.

Experimental procedures used in localizing the C3 epitope and indirectlythe FVIII binding to the 29 kDa polypeptide fragment are explained inmore detail below when these same procedures are used in localizing theC3 epitope and indirectly the FVIII binding to the 170 kDa polypeptidefragment.

The purification of FVIII from commercial factor VIII concentrate(Armour Pharmaceutical, Kankakee, Ill.), by immunoadsorbentchromatography with monoclonal anti-vWF antibody is described in Fulcheret al., Proc. Natl. Acad. Sci. USA 79, 1648-1652 (1982). FVIIIpreparations obtained by this method and used in the followingexperiments had specific activities of 2900-3800 units/mg. Purified vWFwas obtained from commercial factor VIII concentrate (ArmourPharmaceutical, Kankakee, Ill.), by immunoadsorbent chromatography witha monoclonal anti-vWF antibody bound to Sepharose as described inFulcher et al. The bound vWF was eluted by 3M NaSCN as described inFujimara et al., J. Biol. Chem. 261, 381-385 (1986) and concentrated anddesalted with a tangential flow Minitan ultrafiltration system(Millipore, Bedford, Mass.), with a 100,000 molecular weight cut offmembrane. The protein was further dialyzed extensively against 0.05MTris, 0.15M NaCl, pH 7.35 (TBS).

Mouse monoclonal anti-FVIII and anti-vWF antibodies were produced,purified, and characterized and described in Fulcher et al., andFujimara et al. Radioiodination of monoclonal anti-FVIII and anti-vWFantibodies were done according to the method of Fraker and Speck,Biochem. Biophys. Res. Commun. 80, 849-857 (1978), to a specificactivity of 3-10×10⁹ cpm/mg.

SP fragment-III was obtained by limited proteolysis of vWF withStaphylococcus aureus V8 protease (Sigma, St. Louis, Mo.), and purifiedby the method of Girma et al., Biochemistry 25, 3156-3163 (1986), withmodifications as described by Titani et al., Biochemistry 25, 3171-3184.All fragments were dialyzed against TBS pH 7.35 before testing.

The reduction and alkylation of vWF was performed as has been previouslydescribed in Fujimara et al.

Two dimensional crossed immunoelectrophoresis of vWF was performed asdescribed in Zimmerman et al., Immunoassays: Clinical LaboratoryTechniques for the 1980's, pp. 339-349, Alan R. Liss, Inc., New York(1980), with the following modifications. Agarose was poured in a 1.5 cmstrip at the bottom of a 10.2 cm×8.3 cm piece of Gelbond (FMCCorporation, Rockland, Me.). Purified vWF or fragments of vWF, FVIII,and ¹²⁵ I-labeled monoclonal anti-FVIII antibody were mixed in thesample well and electrophoresed. A second gel containing 125-250 μl ofrabbit serum containing polyclonal anti-vWF antibodies was then pouredand the second dimension was electrophoresed at right angles to thefirst dimension. Autoradiographs were made of the gels and compared toCoomassie brilliant blue staining of the gels.

Competitive inhibition assay of FVIII binding to solid phase vWF: 50 μgof whole unreduced vWF in 1 ml of 0.01M PO₄, 0.15M NaCl, 0.02% NaN₃, pH7.3 (PBS), was incubated with three 1/4 inch in diameter polystyrenebeads (Pierce Chemical Company, Rockford, Ill.) per 16 mm in diametertissue culture well for 2 hours at room temperature. The solution wasremoved and the wells and the beads were then blocked with 1 ml of PBScontaining 0.05% Tween-20 and 3% human serum albumin for 1 hour at roomtemperature. The wells and the beads were stored in the blockingsolution at 4° C. for 16 hours to 10 days before use. The wells andbeads were then washed ×3 with PBS 0.05% Tween-20 and incubated for 11/2hours at room temperature with 1.3 μg of purified FVIII and 0-100 μg ofthe competitive ligand in 1 ml of 0.05M imidazole, 0.15M NaCl, 0.02%NaN₃, pH 7.0, 3 mM CaCl. The beads then were washed ×5 with PBS 0.05%Tween-20 and incubated for 11/2 hour at room temperature with 1.5×10⁶cpm of ¹²⁵ I-monoclonal anti-FVIII antibody C2 (specific activity3.8×10⁹ cpm/mg), in 1 ml of PBS 0.05% Tween-20 containing 0.5% bovinegamma globulin. After incubation, the wells and beads were washed withPBS 0.05% Tween-20×2. The beads were then transferred to clean wells andwashed an additional four times and separately counted. Total cpm in theabsence of competing ligands ranged from 1340-2520 cpm in differentexperiments Background counts were those obtained when ¹²⁵ I-monoclonalanti-FVIII antibody C2 was incubated with the vWF coated beads in theabsence of FVIII. These ranged from 60-200 cpm

Protein concentrations were determined by the method of Bradford, Anal.Biochem 72:248-254 (1976), using bovine serum albumin as a standard.

Crossed immunoelectrophoresis demonstrated complex formation betweenpurified vWF and purified FVIII. This was shown by co-precipitation of¹²⁵ I-labeled monoclonal anti-FVIII antibody with unlabeled vWF onlywhen purified FVIII was included in the sample well. In order tolocalize the FVIII binding domain, similar experiments were performedwith vWF fragments obtained by Staphylococcus aureus V8 proteasedigestion. Limited digestion of vWF with Staphylococcus aureus V8protease has been reported to produce primarily a single cleavage in vWFyielding two major fragments. SP fragment II is a 110-kDa homodimercontaining the carboxy-terminal portion of the vWF molecule (residues1366-2050) and SP fragment III is a 170-kDa homodimer containing theamino-terminal portion of the vWF molecule. This 170-kDa polypeptidefragment has an amino-terminal sequence beginning with amino-terminalamino acid residue 1 Ser and a carboxy-terminal amino acid residueextending no further than amino acid residue 1365-Glu according to theamino acid sequence published in Titani et al., Biochemistry, 25,3171-3184 (1986). These two fragments represent 100% of the molecularmass of the vWF subunit. Complex formation was demonstrated betweenFVIII and the amino-terminal SP fragment III but not with thecarboxy-terminal SP fragment II. This indicates that the amino-terminalSP fragment III in its homodimer form maintains the capability ofinteraction with FVIII in a qualitatively similar way as that of wholevWF. The carboxy-terminal SP fragment II in its homodimer form does notdemonstrate this FVIII binding capability.

The monoclonal anti-vWF antibody C3 largely inhibited complex formationbetween FVIII and vWF when it was included in the sample well, whereas80 other monoclonal anti-vWF antibodies (tested in pools of 5 each) werewithout effect. C3 also inhibited complex formation between FVIII and SPfragment III in this system. Direct reactivity of C3 with SP fragmentIII was shown by adding ¹²⁵ I-labeled C3 to a sample well containingpurified SP fragment III. Autoradiographs of the crossedimmunoelectrophoresis gel showed co-precipitation of the radiolabeledantibody with SP fragment III. In a similar experiment, noco-precipitation with SP fragment II occurred.

In order to better characterize FVIII binding to vWF, a competitiveinhibition assay was developed. In this assay purified vWF or SPfragment III was adsorbed to the surface of polystyrene beads. The beadswere then incubated with purified FVIII. Purified FVIII bound to bothunreduced vWF and unreduced SP fragment III which had been immobilizedon the surface of the polystyrene beads. This was demonstrated by thebinding of ¹²⁵ I-labeled monoclonal anti-FVIII antibody to polystyrenebeads sequentially incubated with vWF and FVIII.

Both the binding of FVIII to vWF and the binding of ¹²⁵ I-labeledmonoclonal anti-FVIII antibody to FVIII were specific in this system asdemonstrated by the following experiments. First, the binding of FVIIIwas shown to be dependent on the presence of vWF adsorbed to the surfaceof the polystyrene beads. When the polystyrene beads were coated withhuman serum albumin and then incubated with FVIII, followed by ¹²⁵I-labeled monoclonal anti-FVIII antibody, the counts per minute measuredwere only 2% of that seen with FVIII binding to vWF coated polystyrenebeads. Secondly, when vWF coated polystyrene beads were not incubatedwith FVIII, the bead associated counts per minute were only 1% of thatseen when the FVIII incubation was included.

The reversibility of the binding of FVIII to the immobilized vWF couldalso be demonstrated. Dissociation of FVIII from the vWF-FVIII complexhas been shown to occur in the presence of 0.25M CaCl₂ according toCooper et al., J. Clin. Invest. 54, 1093-1094 (1974), 10-20 mM EDTAaccording to Tran et al., Thromb. Haemostas. 50, 547-551 (1983) or1-1.5M NaCl according to Weiss et al., Thromb. Diath. Haemorrh. 27,212-219 (1972). In the polystyrene bead system, five washings of thepolystyrene beads with an imidazole buffered saline containing 0.25MCaCl₂ at 37° C. produced 70±4% dissociation of FVIII from vWF.Similarly, five washings with an imidazole buffered saline containing 20mM EDTA produced 66±5% dissociation and with an imidazole buffercontaining 1.5M NaCl produced 86±1% dissociation of FVIII from vWF. Fivewashings with the same imidazole buffered saline containing 3 mM CaCl₂produced no FVIII dissociation from vWF adsorbed to the polystyrenebeads.

The specificity of the binding of fluid phase FVIII to vWF immobilizedto the surface of the polystyrene beads was also shown by the ability ofwhole, unreduced vWF in fluid phase to completely inhibit this binding.Reduced and alkylated vWF had no inhibitory effect on FVIII binding.Reduced and alkylated vWF, and reduced and alkylated SP fragment III,were also unable to bind FVIII in the crossed immunoelectrophoresissystem. These findings are consistent with the observation that undermild reducing conditions FVIII can be dissociated from vWF, see Blombacket al., Thromb. Res. 12, 1177-1194 (1978).

SP fragment III demonstrated dose dependent inhibition of FVIII bindingwith 90% inhibition at a concentration of 50 μg/ml. SP fragment I, aproduct of Staphylococcus aureus V8 protease digestion of SP fragmentIII which contains the middle portion of the vWF molecule (residues911-1365 as described in Titani et al., Biochemistry 25, 3171-3184(1986)) produced only 15% inhibition at concentrations up to 100 μg/ml.These data localized a major FVIII binding domain to the amino-terminalportion of vWF. SP fragment II inhibited FVIII binding by 29% at aconcentration of 50 μg/ml. Doubling the concentration produced nosignificant increase in inhibition.

The complete 2050 amino acid sequence of vWF has been determined byprotein sequence analysis, see Titani et al., Biochemistry 25, 3171-3184(1986). With such information a nucleotide sequence can be inserted intothe appropriate vector for expression of the 29 kDa and 170 kDapolypeptide fragments and sequential subsets of polypeptide fragmentswhich inhibit binding of vWF to FVIII. For a description of recombinantDNA techniques for cloning vWF fragments, see Ginsburg et al., Science228:1401-1406 (1985) and Sadler et al., Proc. Nat. Acad. Sci. USA 82,6394-6398 (1985).

Peptides at least three amino acid residues in length beginning from theamino-terminal region of the 29 kDa polypeptide fragment are synthesizedas described by Houghton et al., Proc. Natl. Acad. Sci. USA 82:5135(1985).

In the well known procedure for solid-phase synthesis of a peptide, thedesired peptide is assembled starting from an insoluble support such asbenzhydryl amine or chloromethylated resin (derived from cross-linkedpolystyrene, and available from chemical supply houses). The amino acidat the carboxy-terminal end of the desired polypeptide, carryingprotecting groups on the alpha-amino nitrogen and on any other reactivesites, is attached to the resin from solution using known peptidecoupling techniques. The protecting group on the alpha-amino group isremoved (leaving other protecting groups, if any, intact), and the nextamino acid of the desired sequence (carrying suitable protecting groups)is attached, and so on. When the desired polypeptide has been completelybuilt up, it is cleaved from the resin support, all protecting groupsare removed, and the polypeptide is recovered. Examples of suitableprotecting groups are: alpha-tert-butyloxycarbonyl for thealpha-amino-group; benzyl, 4-methoxybenzyl, or 4-methylbenzyl for thethiol group of cysteine, the beta-carboxylic acid group of asparticacid, the gamma-carboxylic acid group of glutamic acid and the hydroxylgroups of serine, threonine, and tyrosine; benzyloxycarbonyl or a2-chloro- or 3, 4-dimethoxy-derivative thereof for the ring nitrogens ofhistidine and tryptophan and the epsilon-amino group of lysine;p-nitrophenyl for the amide nitrogens of asparagine and glutamine; andnitro or tosyl for the guanidine group of arginine.

For purposes of this disclosure, accepted short-hand designations of theamino acids have been used. A complete listing is provided herein below:

One and Three-letter Amino Acid Abbreviations

    ______________________________________                                        A       Ala      Alanine                                                      C       Cys      Cysteine                                                     D       Asp      Aspartic Acid                                                E       Glu      Glutamic Acid                                                F       Phe      Phenylalanine                                                G       Gly      Glycine                                                      H       His      Histidine                                                    I       Ile      Isoleucine                                                   K       Lys      Lysine                                                       L       Leu      Leucine                                                      M       Met      Methionine                                                   N       Asn      Asparagine                                                   P       Pro      Proline                                                      Q       Glu      Glutamine                                                    R       Arg      Arginine                                                     S       Ser      Serine                                                       T       Thr      Threonine                                                    V       Val      Valine                                                       W       Trp      Tryptophan                                                   Y       Tyr      Tyrosine                                                     B       Asx      Asp or Asn, not distinguished                                Z       Glx      Glu or Gln, not distinguished                                X       X        Undetermined or atypical amino acid                          ______________________________________                                    

One or more of the peptides of the present invention can be formulatedinto pharmaceutical preparations for therapeutic, diagnostic, or otheruses. To prepare them for intraveneous administration, the compositionsare dissolved in water containing physiologically compatible substancessuch as sodium chloride (e.g. 0.35-2.0M), glycine, and the like andhaving a buffered pH compatible with physiological conditions. Theamount to administer for the prevention of thrombosis will depend on theseverity with which the patient is subject to thrombosis, but can bedetermined readily for any particular patient.

The following example is given as illustrative of the present invention.The present invention is not restricted only to this example.

EXAMPLE 1 Preparation of monoclonal antibody, C3, from hybridoma cellline

In the procedure for production of the hybridoma cell line producingmonoclonal anti-vWF antibody C3 mice of strain BALB/c (ResearchInstitute of Scripps Clinic) were immunized intraperitoneally withpurified FVIII immunogen containing small amounts of vWF whichco-purified with it as a contaminant. The FVIII was prepared asdescribed in Fulcher et al., Proc. Natl. Acad. Sci. USA 79, 1648-1652(1982). The mice were immunized intraperitoneally with 1 μg of immunogenin complete Freund's adjuvant. Seven days later the mice were immunizedintraperitoneally with 10 μg of immunogen in incomplete Freund'sadjuvant. Seven days after this second injection they were immunizedintraperitoneally with 50 μg of immunogen in incomplete Freund'sadjuvant. Eight days after this third injection they were immunizedintraperitoneally with 100 μg of soluble immunogen. Spleens were removedthree days later, and spleen cells were fused with P3×63-AG8.653 (mousemyeloma cell line).

P3X653-AG8.653 was maintained (before fusion) at log phase growth in amedium of 90% Dulbecco's modified Eagle's medium (high glucose) and 10%Fetal bovine serum (FBS). The following recommended supplements wereadded to 475 ml of the above medium: glutamine (100x) 5 ml, sodiumpyruvate (100x) 5 ml, nonessential amino acids (100x) 5 ml,Pen-strep-fungizone (100x) 5 ml and 8-azaguanine 6.6×10⁻³ M (50x) 10 ml.Spleen and myeloma cells were washed thoroughly without FBS inDulbecco's modified Eagle's medium before fusion. Cells were fused with1 ml 40% PEG 1500 for 1 minute. Then cells were diluted 1:2 with growthmedium for 1 minute. Cells were diluted further 1:5 with growth mediumfor 2 minutes. Next cells were spun 900 RPM for 10 minutes. Thesupernatant was removed, the cells were selected by suspension in HATmedium and placed in 96 well plates. The Hat medium contained 90%Dulbecco's modified Eagle's medium (high glucose), 10% FBS and thefollowing recommended supplements added to 405 ml of the above twocomponents: glutamine (100x) 5 ml, NCTC 109 50 ml, sodium pyruvate(100x) 5 ml, nonessential amino acids (100x) 5 ml, Pen-strep-fungizone(100x) 5 ml, (hypoxanthine 10⁻² M+thymidine 1.6×10⁻³ M) (100x) 5 ml,bovine insulin (20 I.U./ml)(100x) 5 ml, oxaloacetate (10⁻¹ M) (100x) 5ml and aminopterin (2×10⁻⁵ M)(50x) 10 ml. For 4 weeks followingselection the cells were maintained in growth medium--HT (selectionmedium minus aminopterin). Subcloning was accomplished by limitingdilution. Wells with growth are tested by ELISA assay. Test plates werecoated with 100 ng/well immunogen or human fibrinogen, or humanfibronectin, or human vWF, each protein being a potential contaminant ofthe immunogen. 50 μg of culture supernatant were tested. Those wellscontaining cells whose supernatants were positive with a vWF were grownat 37° C. in 10% CO₂.

For ascites production the mice were primed with 0.5 ml pristine atleast 4 days before cell injection. The cells were injectedintraperitoneally (5×10⁶ /mouse) in 0.5 ml media with on FBS. Theascites were harvested when the mice bloated. The monoclonal anti-vWFantibody C3 contained in the mouse ascites is of the IgG-1 type.

The following Protein A sepharose purification of monoclonal anti-vWFantibody C3 from mouse ascites is a modified procedure of that disclosedin Ey et al, Immunochemistry, 15, 429-436 (1978). The amounts used werefor a column 1 cm×15 cm which bound about 25-30 mg IgG-1, but whichallowed separation of about 50 mg IgG-1 from non IgG proteins. Thecolumn can also bind 50 mg of IgG2a. IgG2b also binds to the column, butIgM, IgA and IgE do not bind. 4-6 ml of ascites was centrifuged at30,000 rpm for 45 minutes. The lipids were removed on top. The additionof 20% sucrose weight/column to the ascites aided in the removal oflipids. Ascites was diluted to 25-30 ml with 140 mM NaPO₄ buffer, pH8,containing 0.02% NaN₃. The ascites was diluted to prevent theinterference of chloride ion with the binding of IgG. Approximately 2 gof Protein A sepharose (Sigma) was swollen in 10 mM phosphate bufferedsaline with 0.02% NaN₃ and packed into a 1 cm diameter column. Thecolumn was equilibrated in 140 mM NaPO₄ buffer with 0.02% NaN₃. Thecolumn was loaded with diluted ascites at 0.06-0.03 ml/min or less. Thecolumn was allowed to sit at 4° C. overnight after loading to increasebinding of IgG. The column was washed with buffers at 0.6-0.8 ml/min inthe following order:

1) 140 mM NaPO₄, pH 8.0; 2) 0.1M Na citrate-citric acid, pH 6.0 (IgG-1eluted); 3) 0.1M Na citrate-citric acid, pH 5.0--IgG2a eluted and asmall percentage of remaining IgG-1; 4) 0.1M Na citrate-citric acid(small percentage of remaining IgG2a eluted); and 5) 0.1M Nacitrate-citric acid, pH 3.0 (IgG2b eluted). As soon as the column waswashed with pH 3.0 buffer, it was washed with 140 mM NaPO₄ buffer, pH8.0+0.02% NaN₃ until pH of effluent is 8.0. The column was stored at 4°C. During the washing of the column approximately 5 ml fractions werecollected. To any fraction of pH 5.0, 1 ml of 1M tris HCl was added.

What is claimed is:
 1. A 29 kDa von Willebrand Factor polypeptide havingan amino acid sequence which is a sequential subset of the followingsequence: ##STR3## wherein the N-terminal amino acid of said polypeptideis selected from amino acid 3 (Ser) through 44 (Gly) of said sequenceand the C-terminal amino acid of said polypeptide is selected from aboutamino acid 244 (Leu) through 285 (Asp) of said sequence, saidpolypeptide further characterized by its ability to inhibit binding ofvon Willebrand Factor to Factor VIII.
 2. A polypeptide according toclaim 1 wherein said polypeptide has the amino-terminal sequencebeginning with amino-terminal amino acid residue 3 Ser and endingapproximately with carboxy-terminal amino acid residue 244 Leu.
 3. Apolypeptide according to claim 1 wherein said polypeptide has theamino-terminal sequence beginning with amino-terminal amino acid residue24 Glu and ending approximately with carboxy-terminal amino acid residue265 Ser.
 4. A polypeptide according to claim 1 wherein said polypeptidehas the amino-terminal sequence beginning with amino-terminal acidresidue 44 Gly and ending approximately with carboxyl-terminal aminoacid residue 285 Asp.
 5. A von Willebrand Factor polypeptide having theamino acid sequence: ##STR4##
 6. A peptide of claims 1, 2, 3, 4, or 5further characterized by binding with the monoclonal anti-vWF antibodyC3 secreted by the hybridoma ATCC Designation No. HB9425 capable ofspecifically binding to the region of von Willebrand Factor containingthe Factor VIII binding domain.